The long term objective of our research is to understand the regulatory mechanisms of proliferation and differentiation of normal and neoplastic mammary epithelial cells. Mammalian cells in culture can be classified as ethanolamine (Etn)-responsive or - nonresponsive with regard to their growth. Normal mammary cells are Etn-responsive and some neoplastic mammary cells are also Etn- responsive, whereas other neoplastic mammary cells are Etn- nonresponsive. In Etn-responsive cells de novo biosynthesis of phosphatidylethanolamine (PE) is limited due to the reduced activity of the serine exchange enzyme which is responsible for the biosynthesis of the precursor of PE, phosphatidylserine (PS). When the medium is not supplemented with Etn, the membrane phospholipid in Etn-responsive cells becomes PE-deficient, the proliferation stops. The immediate objective is to elucidate the biochemical mechanism of Etn-responsive and -nonresponsiveness of mammary cells and to identify the gene or genes responsible for conferring to the cells Etn-nonresponsiveness. The present proposal has three specific aims. The first aim is to elucidate the cause of the reduced activity of the serine exchange enzyme in Etn-responsive cells. The reduced activity of the enzyme does not seem to result from a limited amount of enzyme, but rather results from an actual reduction in activity. The properties of the enzyme will be examined using crude cell extract and the microsomal fraction. The second aim is to analyze the effect of PE-deficiency on extracellular signal transductions in order to elucidate the mechanism of cessation of cell proliferation due to PE-deficiency. We already have clear indications that a signal transduction mediate by tumor promoting phorbol esters and EGF receptor does not proceed properly. Further analyses of binding characteristics of EGF and insulin will be carried out. The third aim is to identify and isolate the gene (or genes) which renders the cell Etn-nonresponsive. This aim will be accomplished by 1) studying int-1 and int-2 oncogenes which have initially been shown to convert Etn-responsive cells to -nonresponsive, and 2) isolating DNA fragment from an Etn-nonresponsive human breast carcinoma cell line by transfection of Etn-responsive rat carcinoma cells. These studies will contribute to the understanding of the role of membrane phospholipids in various cellular metabolic processes and the process of malignant tumor progression.